Dimerization mechanisms of two procaspase subfamilies

描述（由申请人提供）： 调节前半胱氨酸蛋白酶激活的机制在细胞凋亡和炎症的调节中起着核心作用。已经显示，半胱天冬酶原二聚体的形成是成熟中的关键事件。例如，半胱天冬酶原-1被认为是单体，直到半胱天冬酶募集结构域（CARD）中的相互作用驱动蛋白酶结构域的二聚化。支架足以允许自溶处理。相比之下，我们已经表明，procaspase-3是一个稳定的二聚体，即使它不包含CARD。这表明不同的折叠和调节机制的活化的前半胱氨酸蛋白酶-1和-3。我们推测，二聚体界面的差异是决定蛋白质是单体还是二聚体的关键。此外，我们表明，二聚化和酶活性的联系。基于我们的蛋白质工程研究，我们假设蛋白质稳定性和酶活性的增加是通过从二聚体界面延伸到两个活性位点的氨基酸网络连接的。我们认为，procaspases作为分子机器中的侧链运动在二聚体接口的影响，在活性位点的运动，允许底物结合口袋的形成。我们将通过解决以下具体目标中的三个关键问题来解决这个问题。1.前半胱氨酸天冬氨酸蛋白酶-1和-3是否通过类似的机制折叠和组装？将采用已建立的生物物理方法来确定半胱天冬酶原-1的寡聚体性质，以测试当前的范例。2.二聚化与活性位点的形成有何联系？使用蛋白质工程技术，我们将研究在四个位置附近的二聚体接口和活性位点，影响正确插入的活性位点环的氨基酸的明显的连接。3.前结构域在折叠和组装中如何发挥作用？证据表明，前肽功能作为分子内伴侣。将采用分子生物学和生物物理学研究来确定确切的作用机制。这项工作有可能影响一些自身免疫性疾病，leart疾病和癌症的治疗策略，因为细胞凋亡是这些疾病的共同因素。学习选择性地操纵细胞凋亡的水平可能会导致这些疾病的治疗策略。

DESCRIPTION (provided by applicant): The mechanisms that regulate the activation of procaspases play central roles in the regulation of apoptosis and inflammation. It has been shown that formation of a procaspase dimer is a critical event in maturation. For example, procaspase-1 is thought to be a monomer until interactions in the caspase recruitment domain (CARD) drive dimerization of the protease domains. The scaffold is sufficient to allow autolytic processing. In contrast, we have shown that procaspase-3 is a stable dimer, even though it does not contain a CARD. This suggests different folding and regulatory mechanisms for the activation of procaspases-1 and -3. We hypothesize that differences in the dimer interfaces are the key to whether the protein is a monomer or dimer. In addition, we show that dimerization and enzymatic activity are linked. Based on our protein engineering studies, we hypothesize that the gains in protein stability and enzyme activity are linked via a network of amino acids that extends from the dimer interface to the two active sites. We suggest that procaspases act as molecular machines in which side chain movements in the dimer interface affect the movements in the active site, allowing the substrate-binding pocket to form. We will approach this problem by addressing three key questions in the following specific aims. 1. Do procaspases-1 and -3 fold and assemble via similar mechanisms? Established biophysical methods will be employed to determine the oligomeric properties of procaspase-1 in order to test the current paradigm. 2. How is dimerization linked to active site formation? Using protein engineering techniques, we will examine the apparent linkage of amino acids at four positions near the dimer interface and active sites that affect proper insertion of the active site loops. 3. How does the pro-domain function in folding and assembly? Evidence is presented that the pro-peptide functions as an intramolecular chaperone. Molecular biological and biophysical studies will be employed to determine the precise mechanism of action. This work has the potential to affect therapeutic strategies for a number of autoimmune diseases, leart disease, and cancers because apoptosis is a common factor to these diseases. Learning to electively manipulate the level of apoptosis may well lead to therapeutic strategies for these diseases.